CC (midterms) Flashcards
Enzymes
Biologic proteins that catalyze the biochemical reactions
Intracellular proteins to hasten the chemical reaction
Affects the reaction of organic matter
Enzymes are not consumed nor altered
Only the substrates changes the form (substrate to product)
Increased enzymes in the serum may be because:
Cell injury or cell degradation
Increased membrane permeability allowing for proteins to move out of the cell
Function of enzymes:
Hydration of carbon dioxide during respiration
Nerve induction
Muscle contraction
Nutrient degradation
Growth and reproduction
Energy storage and use
LIVER ENZYMES
Aspartate aminotransferase (AST)
Alanine aminotransferase (ALT)
Gamma-Glutamyl transferase (GGT)
Alkaline phosphatase (ALP)
Acid phosphatase (ACP)
MI Profile
Creatine Kinase (CK)
Aspartate Aminotransferase (AST)
Lactate Dehydrogenase (LDH)
Pancreatic Enzymes
Amylase (AMS)
Lipase (LPS)
Prostate enzymes
Acid phosphatase (ACP)
Glucose-6-phosphate dehydrogenase (G6PDH)
Miscellaneous enzymes
- 5’ Nucleotidase (5’N)
- Cholinesterase / Pseudocholinesterase
- Angiotensin-Converting enzyme (ACE)
- Ceruloplasmin
- Ornithine carbamoyl Transferase (OCT)
- Glucose-6-phosphate dehydrogenase (G-6PD)
other name for AST
Serum glutamic-oxaloacetic transaminase or Serum glutamate-oxaloacetate transaminase (SGOT)
Transfer of an amino group between aspartate and a-keto acids
AST
Involved in the synthesis and degradation of AA (amino acids)
AST
Widely distributed, highest activities in cardiac, liver and skeletal muscle
AST
2 isoenzymes of AST
Cytoplasmic
Mitochondrial
most abundant, the most predominant AST in healthy human serum
Cytoplasmic AST
present in the mitochondrial membrane. An increase in this isoenzyme indicates possible necrosis or severe damage of the cell.
Mitochondrial AST
After MI, AST levels begin to rise in ___, peak at ___, and return to normal in ____.
6–8 hours; 24 hours; 5 days
AST increased in
in hepatocellular and skeletal muscle disease
Uses malate dehydrogenase and monitors decrease in absorbance at 340 nm
Karmen Method
method that is falsely ↑ in hemolyzed sample
Karmen Method
Karmen Method reference range:
5-30 U/L
Aspartic acid + a-ketoglutaric acid ⇆ ____________
glutamic acid + oxaloacetic acid
oxaloacetate + NADH + H ⇆ _____
malate + NAD
are enzymes with similar catalytic activity but differ in the physical, biochemical and immunologic properties.
Isoenzymes
a type of cofactor that serves as the second substrate for enzymes.
Coenzymes
When a coenzyme is tightly bound to an enzyme, it is called a ____
prosthetic group
Oxidized form: NAD measures for
Decreased absorbance
Reduced form: NADH
high/increased absorbance
other name for Alanine aminotransferase (ALT)
serum glutamic-pyruvic transaminase (SGPT)
Transfer of an amino group between alanine and a-ketoglutarate
ALT
more liver specific compared to AST
ALT
Increased in hepatocellular disorders
ALT
Significant in the evaluation of hepatic disorders
Markedly increased in acute inflammatory conditions of the liver
Used to monitor the course of hepatitis treatment and the effects drug therapy
Used for screening for post transfusion hepatitis
Used to screen blood donors, (ALT increased with people with jaundice)
Used as sensitive test for occupational toxic exposure
Monitors course of hepa treatment and possible effects of drug therapy
uses of ALT
The AST/ALT Ratio
Differentiates the cause of hepatic disorder
De Ritis Ratio
Ratio > 1
non viral origin
Ratio < 1
viral in origin
pyruvate + NADH + H ⇆ _____
Lactate + NAD
pyruvate +glutamate ⇆ _____
alanine + a-ketoglutarate
Assay for enzyme activity ALT
Uses Lactate Dehydrogenase and monitors decrease in absorbance at ___
340 nm
COLOR DEVELOPER ALT and AST
2,4 DNPH
COLOR INTENSIFIER ALT & AST
0.4 N NaOH
METHODS for ALT and AST
Reitman and Frankel
Catalyze the transfer of the y-glutamyl residue from y-glutamyl peptides to amino acids H2O, etc
Gamma-Glutamyl transferase (GGT)
In the biologic system, the common donor for y-glutamyl is _____
glutathione
glutathione + amino acid → ___
glutamyl-peptide + L-cysteinyglycine
Used for diagnosis hepatobiliary disorders and chronic alcoholism
GGT
GGT is located in the ____ of the hepatic cells. Specifically in the epithelial cell linings of _____
canaliculi; biliary ductules
Sensitive marker for ethanol intoxication.
Marker for occult alcoholism
GGT
Most sensitive marker for acute alcoholic hepatitis
GGT
Assay for Enzyme Activity in GGT
Szaz Assay
The absorbance of p-Nitroaniline is measured at
405-420 nm
y-glutamyl-p-nitroanilide + glycylglycine → ____
y-glutamyl-glycylglycine + p-nitroaniline
Involved in the storage of high-energy creatine phosphate in muscle cells
Creatine Kinase (CK) or Creatine Phosphokinase (CPK)
CK is widely distributed, highest activities in _____.
skeletal muscle, heart and brain.
creatine phosphate + ADP → ___
creatine + ATP
Methods of determination for CK
Forward Reaction (Tanzer-Gilvarg)
Reverse Reaction (Oliver-Rosalki)
Optimum pH for Tanzer Gilvarg
9.0
Optimum pH for Oliver-Rosalki
6.8
↓ in absorbance at 340 nm is determined
Forward Reaction (Tanzer-Gilvarg)
↑ in absorbance at 340 nm is determined
Reverse Reaction (Oliver-Rosalki)
Source of Error for CK
Hemolysis
inactivation by light
Physical activity and IM injections
Reference Range of CK (male & female; % of CK-MB)
Male: 15-160 U/L; Female: 15-130 U/L
CK-MB: <6% of total CK
Slowest mobility toward the anode
Major isoenzyme in striated muscle and normal serum
Ck-3/CK-MM/
Muscle type
2nd fastest to migrate toward the anode
Significant quantities are found in heart tissues
CK-2/CK-MB/
Hybrid type
Migrate the fastest toward the anode
Highest concentration in CNS, GI tract and uterus (pregnancy)
CK-1/CK-BB/
Brain type
After MI, CK-MB (>6%) levels begin to rise within ___, peak at ____ and return to normal levels within ___
4-8 hours; 12-24 hrs; 48-72 hrs
It is the first enzyme to elevate after a myocardial infarction
CK
CK is considered a ____ . It is composed of two different monomers. Composed of M and B type
dimeric molecule
Reference Values of CKs
CK-MM: 94-98%
CK-MB: 2-6%
CK-BB: <1%
Other CK Isoenzymes
Macro-CK
CK-BB is complexed with antibodies __
IgG or IgA
CK-MM is complexed with ___
lipoproteins
macro-CK migrate midway between ___
CK-MM and CK-MB
Located in mitochondrial membrane, increase in this indicates cell necrosis or severe damage
Migrates cathodal to CK-MM
Mitochondrial CK (CK-Mi)
Where the activity of CK1, CK2 and CK3 are all measured
It is less diagnostic bc it is not tissue specific
Total CK
This targets a particular tissue
Improves the test specificity thus improving diagnosis
CK isoenzyme
Catalyzed the interconversion of lactic and pyruvic acids
Widely distributed, highest activities in heart, hepatic, skeletal muscle and RBC
Lactate Dehydrogenase (LDH)
Is a tetrameric molecule composed of 4 subunits/monomers of 2 possible forms
LDH
lactate + NAD → ___
pyruvate + NADH + H
Composed of 5 isoenzymes
LDH
temp that decreases the activity of LD
low temp/freezer temp
temp (+storage) that can maintain the activity of LD
If room temp for 2 days
Assay of Enzyme Activity for LDH
Wacker method
Wroblewski La Due
a-hydroxybutyrate dehydrogenase (a-HBD)
Reverse Reaction (Pyruvate → Lactate)
Decrease in absorbance is monitored at 340 nm
Three times faster but more susceptible to substrate exhaustion
Wroblewski La Due
Optimal pH of Wroblewski La Due
7.1 to 7.4
Forward reaction (Lactate → pyruvate)
Increase in absorbance is monitored at 340 nm
Commonly used method in LD
Wacker method
Optimal pH of Wacker method
8.3 - 8.9
Has greater affinity of H subunits
a-hydroxybutyrate dehydrogenase (a-HBD)
Represent LDH-1
a-hydroxybutyrate dehydrogenase (a-HBD)
a-ketobutyrate + NADH + H →
a-hydroxybutyrate
After MI, LD begin to rise within ____, peak at ____ and remains elevated for ___
10-24 hrs; 48-72 hrs; 10 days
Reference range of LD
100-225 U/L
LDH is a __ containing two active sub-units
tetramer
Order ng pinakamataas sa healthy serum
LD 2 > 1 > 3 > 4 > 5
order of LD from most anodic (fastest) to least anodic (slowest)
LD 1 > LD 2 > LD 3 > LD 4 > LD 5
🡹LDH 1 & 🡹CK-MB
AMI
🡹LDH 1 and normal CK-MB
hemolytic anemia
LDH 6 (Alcohol Dehydrogenase) present in px with:
Drug hepatotoxicity
Obstructive Jaundice
Artherosclerotic failure
LDH-1 and LDH-2 tissue involved
Heart, RBC
1 - (MI, hemolytic anemia)
2 - (RI, megaloblastic anemia)
LDH-3
Lung, Spleen, Pancreas (Pulmonary embolism)
LDH-4
Liver (Hepatic injury)
LDH-5
Skeletal Muscle (Skeletal muscle injury)
Catalyze the hydrolysis of various phosphomonoesters at an alkaline pH
Liberate inorganic phosphate from an organic phosphate ester with production of alcohol
Alkaline phosphatase (ALP)
Sources of ALP
Sources: Liver, Bone (Osteoblast), Placenta, Intestine, Renal tissues
Requires Mg2+ activator
For evaluation of hepatobiliary and bone disorders
ALP
Reference Range of ALP
30-90 U/L (adult)
70-220 u/L (0-3 months)
50-260 U/L (3-10 years)
60-295 U/L (10-puberty)
Assay for enzyme activity
in ALP
Bowers and McComb
Based on molar absorptivity of P-Nitrophenol at a pH of 10.2
Absorbance is measured at 405 nm (visible light region)
Bowers and McComb
p-nitrophenyl-phosphate (colorless) → ___
p-nitrophenol + phosphate ion (yellow)
methods with β-glycero-phosphate as substrate
Bodansky
Shinowara
Jones
Reinhart
methods with P-nitrophenyl phosphate as substrate
Bessy, Lowry & Brock
Bowers & McComb
method in which phenyl phosphate as substrate
King and Armstrong
methods where
Inorganic PO4 + Glycerol as end product
Bodansky
Shinowara
Jones
Reinhart
P-nitrophenol (yellow)
as end product
Bessy, Lowry & Brock
Bowers & McComb
end product of King and Armstrong
Phenol
ALP Isoenzymes:
Electrophoresis
Electrophoresis - Arranged by most anodal (fastest)
- Liver ALP - fastest
- Bone ALP
- Placental ALP
- Intestinal ALP
Heat labile fraction
🡹 in bone disease, healing of bone fractures, and physiologic bone growth
Bone ALP
Fastest Isoenzyme and 🡹 in liver disease
Two fractions: Major liver & fast liver (a1) band
Liver ALP
Most heat stable fraction and 🡹 in pregnancy
Can withstand heating at 65degC for 30 mins
Placental ALP
Slowest moving fraction, in blood groups B or O
🡹 in fatty meal consumption and GIT disorders
Intestinal ALP
Chemical inhibition in ALP:
Phenylalanine
Placental, Intestinal (Putang Ina)
Chemical inhibition in ALP:
Levamisole
Bone, Liver (Blood Lust ♥)
Chemical inhibition in ALP:
.03 moral urea
Bone (only one to be inhibited)
ALP isoenzyme differentiation by Heat stability
Placental ALP (most stable)
Intestinal ALP
Liver ALP
Bone ALP (least stable)
Total ALP elevations by liver or bone ALP is differentiated by heating of serum at ___
56degC for 10 mins
ALP residual activity is ⬇ decrease to >20%
Liver ALP
ALP residual activity is ⬇ decrease to <20%
Bone ALP
Carcino Placental ALP
Regan ALP:
Nagao ALP
Carcino Placental ALP Inhibitors:
phenylalanine, L-leucine
Most heat stable ALP
Lung, Breast, and Gynecological cancers, bone ALP co-migrator,
Regan ALP
Adenocarcinoma of the pancreas and bile duct, pleural cancer
Nagao ALP
INHIBITED BY:
Phenylalanine
L-leucine
Nagao
INHIBITED BY:
Phenylalanine
Regan
Catalyze the hydrolysis of various phosphomonoesters at an acid pH
Liberate inorganic phosphate from an organic phosphate ester with production of alcohol
Acid phosphatase (ACP)
Acid phosphatase is present in the ff tissue source:
Prostate
Red blood cells
Platelets
Bone
For evaluation of metastatic carcinoma of prostate
Forensic investigation of rape
ACP
inhibits specific prostatic ACP
L-tartrate ions
Reference Range: Prostatic ACP:
0-3.5 ng/ml
Quantitative end point substrate
Thymolphthalein monophosphate
Continuous monitoring substrate
α-napthyl phosphate
Gutman and Gutman substrate and end product
Phenyl PO4;
Inorganic phosphate
Shinowara substrate and end product
PNPP;
P-Nitrophenol
Babson, Read, and Phillips substrate and end product
Alpha naphtyl PO4;
Alpha naphthol
Roy and Hillman substrate and end product
Thymolphthalein MonoPO4
Free thymolphthalein
where pancreatic enzymes r produced
acinar cells of the pancreas
An example of HYDROLASE
Promotes breakdown of starch and glycogen via alpha, 1-6 branching linkages
Amylase (AMS)
Major tissue source of AMS
Pancreas
Salivary gland
Minor tissue source of AMS
Adipose tissues
Fallopian tube
Small intestine
Skeletal muscle
Increased in acute pancreatitis, renal failure, and parotitis
Is the smallest enzyme
The first enzyme to increase in acute pancreatitis
AMS
Amylase increases after ____, the peak is after ___. The level persists for .
2-12 hours; 24 hours; 3-5 days
Salivary amylase - __
ptyalin (fast moving)
Pancreatic amylase -__
amylopsin (slow moving)
The activity of amylase is inversely proportional with the absorbance
Measures the disappearance of starch substrate
Starch-Iodine complex (dark-blue) 🡺 Decrease in color intensity (decreased absorbance)
Amyloclastic
Measures the appearance of the product
Starch 🡺 reducing sugars
Directly proportional of amylase activity with the absorbance
Saccharogenic
Measures the increasing color from production of product-chromogenic dye fragment
Insoluble starch-dye 🡺 double starch-dye fragments
Amylase activity is directly proportional to the starch-dye fragment formation
Chromogenic
Coupling of several enzyme systems to monitor amylase activity
The end product is the 5,6-phosphogluconolactone + 5 NADH (🡹 absorbance)
Continuous monitoring
maltopentose → ___
maltrotriose + maltose
Not excreted and is reabsorb in the plasma
Presence of macroamylase in the plasma
Amylase with antibodies
Macroamylasemia
Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids
Hydrolysis of dietary triglycerides in the intestine to 2-monoglyceride and fatty acids
Lipase (LPS)
Specific for pancreatitis
But in time of elevation, amylase is faster than ___
LPS
2-monoglyceride + 2 fatty acids → ___
triacylglycerol + 2H2O
Larger molecule and remains longer in circulation (7 days)
LPS
Assay for enzyme activity LPS
Estimation of liberated fatty acids
Measurement of the amount of light blocked by the suspension or particles in soluble agents in the sample.
Turbidimetric methods
method where phenolphthalein as indicator for LPS
Cherry Crandall
Tietz indicator
Thymolphthalein + Veronal
end color of Cherry Crandall
Pink
end color of Tietz
Blue
Cherry Crandall & Tietz’s substrate
50% olive oil (triolein)
titrating agent for Cherry and Tietz
0.4N NaOH
end point of Cherry and Tietz
Fatty acid (Oleic acid)
A phosphoric monoester hydrolase
Marker for hepatobiliary disease and lesions of liver
Reference value: 0-1.6 units
5’ Nucleotidase (5’N)
Used as a marker for insecticides/pesticides poisoning
Index of parenchymal function
Cholinesterase / Pseudocholinesterase
Used to monitor the effect of muscle relaxants (succinylcholine) after surgery
Reference value: 0.5 - 1.3 Units (plasma)
Cholinesterase / Pseudocholinesterase
Used as a possible indicator of neuronal dysfunction (ptx w/ alzheimer’s disease)
Conversion of Angiotensin 1 to 2 happens in the lungs
Angiotensin-Converting enzyme (ACE)
A.K.A. Peptidyl Dipeptidase A or Kininase II
Increased in: Sarcoidosis, Acute and Chronic Bronchitis, and Leprosy
Angiotensin-Converting enzyme (ACE)
main source of ACE
Main source: Macrophage and Epithelioid cells
Promotes the vasoconstriction of the renal arterioles to promote increase BP
Stimulates the release of aldosterone
Angiotensin II
Copper-carrying protein which acts as a na enzyme
A marker for wilson’s disease (hepatolenticular disease)
Ceruloplasmin
For hepatobiliary diseases
Reference values: 8-20 mU/ml
Ornithine carbamoylTransferase (OCT)
maintains the NADPH in the reduced form in erythrocytes
a newborn screening marker
Glucose-6-phosphate dehydrogenase (G-6PD)
Maintains the NADPH in the reduced form in the erythrocytes
A newborn screening marker
Glucose-6-phosphate dehydrogenase (G-6PD)
Deficiency can lead to drug-induced
Specimen: red cell hemolysate, serum
Reference value : 10-15 U/g Hgb or 1200-2000 mU/mL packed RBC
Glucose-6-phosphate dehydrogenase (G-6PD)
Glucose-6-phosphate dehydrogenase (G-6PD) is found in the
adrenal cortex, spleen, rbc, and lymph nodes
Increased levels of G6PD can be seen in conditions like:
AMI, megaloblastic anemia
inhibits red cell ACP
Formaldehyde & Cupric ions